Wire in the blood: Part II

In Part II, Cyclingnews ' Anthony Tan examines the robustness of the test used to convict Tyler...

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Tyler Hamilton and wife Haven have been a united, outspoken force against his blood doping conviction, most recently appealing his two-year sentence to the sport's highest authority, the Court of Arbitration for Sport in Lausanne, Switzerland.

News feature, November 23, 2005

In Part II, Cyclingnews' Anthony Tan examines the robustness of the test used to convict Tyler Hamilton of homologous blood transfusion, where much of the debate has been centred. Click here to return to Part I.

The homologous BT test

The test that determines whether a rider has transfused someone else's blood to increase his/her own performance, known as homologous blood doping, made its official debut in August 2004 at the Athens Olympic Games. [To see how it works, read 'In the blood'.] It was also the same time Hamilton's A sample returned a positive for homologous blood transfusion.

However, this wasn't known until the final week of the Vuelta a España, where he returned yet another positive for the same offence on September 21. In Athens, the 'B sample' was accidentally frozen and thus rendered unusable, while in Spain, the second sample was confirmed positive, along with team-mate Santiago Perez (the Spaniard was also sentenced for two years for homologous blood transfusion, and did not contest the decision.)

"Should the sporting public believe that two cyclists in the one team who were found to be positive for homologous transfusion are both chimerics? What are the chances?" - Robin Parisotto, principal scientist involved in the development of the EPO blood test implemented at the Sydney 2000 Games and the former manager and senior scientist at the Australian Institute of Sport's (AIS) haematology and biochemistry laboratory, is convinced that Hamilton's defence is flawed

Ever since the announcement and until this day, much debate has been waged over numerous aspects of the test, but it namely centres around four inter-related issues: the test's methodology; the technology - or more precisely, the lack of it - in terms of the qualitative nature of the assessment (Hamilton referred to it as a 'I know it when I see it' type test); the differing amounts of mixed red blood cell populations found in Athens and at the Vuelta; and the lack of a false positive study done before the test was approved and put into use.

a. Methodology

Hamilton said during his hearing in March this year that the methodology used in the homologous blood transfusion test cannot prove a blood transfusion has taken place. Can this be right?

"That is correct," confirmed our UCI medical source. "The test only provides you with the result [that] there is a presence of a double population of blood cells."

This may appear to be a 'guilty before proven innocent' type of test, but one has to ask: "What was a mixed population of blood cells doing inside Hamilton in the first place?" - a question that was duly noted by the independent arbitration panel of the American Arbitration Association (AAA)/North American Court of Arbitration for Sport (CAS) in Hamilton's March 2005 hearing.

Said the panel: "The question that arises for sport is one of the determining the cause of what the results shows. The presence has four possible causes, other than homologous blood transfusion: disease, bone marrow transplant, intra-uterine transfusion or chimera. Then, it is up to the athlete to show his condition his condition is consistent with one of those results; if cannot prove it, then it is a result of homologous blood transfusion."

From the evidence presented at the hearing, the first three were ruled out, leaving only the possibility that he is chimeric - a 1 in 60,000,000 chance. That is, Hamilton's mother had a 'vanishing twin' in her womb at the same time, and although the foetus died, it passed on part of its blood cells to him. Posed senior sports scientist Robin Parisotto: "Should the sporting public believe that two cyclists in the one team who were found to be positive for homologous transfusion are both chimerics? What are the chances?"

"And," stressed our UCI medical source, "nobody has contested the fact that this technique is not reliable in showing the double population of red cells, or a mixed population of red cells."

b. Technology

After the guilty verdict by the AAA/CAS was handed down to Hamilton six weeks later on April 19, 2005, he went to great lengths criticising the procedure used to verify evidence of a mixed red cell population. He wrote on his website: "There was no quantifiable criteria used, but rather a 'I know it when I see it' evaluation."

However, as noted by Parisotto, "The technology which assesses mixed blood cells has been around for over two decades in many pathology laboratories around the world and the detection of mixed blood cells is a routine procedure. The subjective and qualitative 'know it when I see it' type of test has been used when matching donor blood with recipients' blood to exclude mixed blood cell populations, in order to prevent fatal blood reactions."

Also, qualitative assessment isn't new in anti-doping circles within sport, and particularly within cycling. In fact, it's the norm. "Most of the anti-doping tests rely only on the qualitative," said our source from the UCI.

"For stimulants, like anabolics, if it is not a threshold substance, the qualitative result is enough; if it shows the presence of the substance in the urine, that's enough - you don't have to rely on the quantitative [data]. The quantitative is only involved when you have a threshold for that substance, like nandrolone, like ephedrine. In those cases, you have to have a quantification: the qualitative is not enough; you have to say: 'You are under the threshold or you are above the threshold.' But the quantitative is not a requirement for most of the anti-doping tests," he said.

Furthermore, the technique used to assess a 'cross-match' or not - referred to as flow cytometry - is automated to the extent that it quantifies the proportion of mixed (or foreign) cells, so it provides an objective assessment by comparison.

c. Variation in findings

The differing amount of mixed red cell populations found in the two instances where tests provided evidence of homologous blood transfusions can be easily explained, according to medical experts. Its premise is based on the simple notion that due to the average life span of red blood cells (around 120 days), one would expect that over time, the 'foreign' red blood cells would eventually disappear.

"This explains why the initial Athens sample had a higher percentage of mixed cells than in the Vuelta and by February 2005, the mixed population was no longer present as evidenced by Hamilton's counsel expert," said Parisotto.

"Over time," he added, "the concentration of mixed cells will decrease, just like any ingested drug. To infer that the Vuelta samples which had lower values than the Athens sample means that the test does not provide an objective assessment is total nonsense. The survival of transfused blood cells in the circulation is well documented and they eventually disappear from the blood just like drugs. Over a 30 day period, it would be expected that mixed cells would decrease by about 50 percent."

d. False positive study

Perhaps the most intriguing of all - perhaps not - is the lack of a false positive study conducted before the test for homologous blood transfusion was approved, raising a few eyebrows in scientific circles.

No matter how knowledgeable or anally-retentive the scientists are who conduct the test or evaluate the test results, there will always be a margin for error. At the March hearing, the experts speaking for the World Anti-Doping Agency (WADA) simply said they didn't need to do it - not the most scientific of responses. That said, expert medical sources close to Cyclingnews have said the chances of a false positive, such as an error in applying the methodology as noted by one of the Nelson validation studies, is close to zero.

"It also depends what you mean by a false positive," noted the UCI medical source.

"If [the red cells] are different, the presence of a mixed red blood population due to chimera, even if it is remote, is not a false positive, because the tests have shown something consistent. So to give a situation where there is no chimera, there is no medical condition... which could create a mixed population of red cells and the result shows a mixed population of red cells, from what I know, it's almost equal to zero."

In addition, just like any previous doping test before it, the method had to be published in a peer review journal and accepted by the scientific community, it had to be the consensus of a group of experts, and, perhaps most importantly, it had to be reproduced in another laboratory with consistent results. However, the fact remains that highly sensitive screening tests such as those used to detect EPO and homologous blood doping can lead to false positives, and should ideally be followed by a highly specific test to weed out the innocent.

Tyler's next steps

Ever since the Olympic Gold medallist tested positive for blood transfusion on September 11, 2004, through to a guilty verdict handed down by the American Arbitration Association/Court of Arbitration for Sport on April 19 this year - and now to his place of last resort, the Court of Arbitration for Sport (CAS), Hamilton has strenuously maintained his innocence. "Certainly not. I've never transfused blood in my life," he said to Cyclingnews. [see interview: 'Hamilton reacts to doping ban']

Following the AAA/CAS verdict, the 34 year-old American immediately announced his intention to appeal to the sport's governing body on such matters, the Court of Arbitration for Sport (CAS) based in Lausanne, Switzerland, which was filed on May 27, 2005. However, due to the complexity of the issue and number of witnesses involved, the panel went to Hamilton, and not until September 6.

At this stage, the final verdict is still unknown. A CAS statement released after the September hearing said: "The arbitration was adjourned with the agreement that it would be resumed at a later date for the presentation of additional evidence and closing arguments. The date and location of the second hearing have not been fixed yet. The parties have agreed that they would not make any public statements concerning this matter until the final decision of CAS." A subsequent report from news agency Associated Press said the CAS hopes to reach a decision by the end of the year or the beginning of 2006.

A test for autologous transfusion?

With the urinary test for EPO recently put into question after three triathletes were incorrectly identified as having taken cycling's perennial blood-boosting agent [see separate story: 'Serious concerns over urinary EPO test'], there's also the 'loophole' that exists with no approved test for autologous blood transfusion as yet - that is, the transfusion of one's own blood.

At the World Track Championships in Los Angeles, outgoing UCI president Hein Verbruggen indicated that the test to detect doping via autologous blood transfusion was almost complete. However, that was over nine months ago.

"Well, I know people are working on it... it's too soon to say how far they are having the validated test for autologous transfusion," said the UCI medical source. "I'm not involved as much in the development, so I don't know what stage they are at; what I know is that it doesn't exist, but that doesn't mean it couldn't exist in the near future."

While WADA's responsible for coordinating and in large part funding the development of the test, one would think the UCI would play an active role on the sidelines. But from the recent bickering over who leaked Lance Armstrong's doping control reports to French sports paper L'Equipe and the subsequent accusations made by WADA chief Dick Pound to Verbruggen, it's obvious that for time being at least, the UCI are resigned to the role of handing out oranges at half-time.

What is known is that the parameters are modified on a similar level, so the off scores and the reticulocytes behave in the same way, no matter what kind of transfusion occurs. "So when we see a high [off] score or very low reticulocytes, we don't know if it was due to a previous use of EPO, if it due to autologous transfusion, or if it was due to homologous transfusion," said the UCI source, "but we know it was due to blood doping.

"So what this means is that even though autologous transfusion cannot be detected by an anti-doping test, evidence of its use can be seen and the consequences can be already taken, like taking out the athlete from the race, or other measures."